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Claude Gaillardin - One of the best experts on this subject based on the ideXlab platform.
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Molecular cloning of Rab-related Genes in the yeast Yarrowia lipolytica. Analysis of RYL1, an essential Gene encoding a SEC4 homologue
Current Genetics, 1995Co-Authors: Brigitte Pertuiset, Jean-marie Beckerich, Claude GaillardinAbstract:Small GTP-binding proteins of the Rab family are involved in the vesicular traffic inside eukaryotic cells. A Gene Library from the yeast Yarrowia lipolytica was screened with an oligonucleotide deduced from a highly conserved sequence in the Rab family. Four different Genes were isolated. One of them, RYL1 , was shown to be essential for cell viability. RYL1p displayed a high similarity with and tight phyloGenetic relationships to SEC4p. When placed under the control of the GAL10 promoter, RYL1 was able to specifically relieve the thermosensitivity of a sec4–8 mutant of Saccharomyces cerevisiae . Therefore, it is proposed that RYL1 is a functional homologue of the S. cerevisiae SEC4 Gene and is involved in the fusion of secretory vesicles with the plasma membrane in the General protein secretion pathway.
Xunjia Cheng - One of the best experts on this subject based on the ideXlab platform.
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Production of an Anti-Severe Acute Respiratory Syndrome (SARS) coronavirus human monoclonal antibody fab fragment by using a combinatorial immunoglobulin Gene Library derived from patients who recovered from SARS
Clinical and Vaccine Immunology, 2006Co-Authors: Jinye Liu, Yanlin Tao, Gengxi Hu, Brian Cao, Lisheng Qian, Hongxia Shao, Hiroshi Tachibana, Xiaoli Yang, Bin Yang, Xunjia ChengAbstract:A combinatorial human immunoglobulin Gene Library was constructed from the peripheral lymphocytes of two patients who recovered from severe acute respiratory syndrome (SARS). The Library was screened for the production of Fab antibody fragments to a recombinant spike protein of SARS-associated coronavirus (SARS-CoV). One Fab clone, AS3-3, reacted with the spike protein in an enzyme-linked immunosorbent assay. The dissociation constant of AS3-3 was 1.98 x 10(-8) M. Immunofluorescent microscopy revealed that it reacted with SARS-CoV-infected cells. The Library seems to be a potent tool for the production of human antibodies to SARS-CoV.
C. M. Gaillardin - One of the best experts on this subject based on the ideXlab platform.
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Complementation of Saccharomyces cerevisiae acid phosphatase mutation by a genomic sequence from the yeast Yarrowia lipolytica identifies a new phosphatase
Current Genetics, 1992Co-Authors: B. Y. Tréton, M. -t. Dall, C. M. GaillardinAbstract:A Yarrowia lipolytica Gene Library was constructed in vector YRp7 and transformed into a Saccharomyces cerevisiae strain lacking both major acid phosphatase activities. A 2.18 kb genomic sequence restoring the ability to hydrolyze α-naphthyl phosphate was isolated. Its sequencing revealed an ORF encoding 358 amino acids without significant homology with any known phosphatase. A putative signal peptide and several possible sites for N-glycosylation were identified. Phosphate-regulated expression of the cloned Gene was observed in Y. lipolytica . Disruption data favoured the hypothesis that it might encode a minor phosphatase species.
Brigitte Pertuiset - One of the best experts on this subject based on the ideXlab platform.
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Molecular cloning of Rab-related Genes in the yeast Yarrowia lipolytica. Analysis of RYL1, an essential Gene encoding a SEC4 homologue
Current Genetics, 1995Co-Authors: Brigitte Pertuiset, Jean-marie Beckerich, Claude GaillardinAbstract:Small GTP-binding proteins of the Rab family are involved in the vesicular traffic inside eukaryotic cells. A Gene Library from the yeast Yarrowia lipolytica was screened with an oligonucleotide deduced from a highly conserved sequence in the Rab family. Four different Genes were isolated. One of them, RYL1 , was shown to be essential for cell viability. RYL1p displayed a high similarity with and tight phyloGenetic relationships to SEC4p. When placed under the control of the GAL10 promoter, RYL1 was able to specifically relieve the thermosensitivity of a sec4–8 mutant of Saccharomyces cerevisiae . Therefore, it is proposed that RYL1 is a functional homologue of the S. cerevisiae SEC4 Gene and is involved in the fusion of secretory vesicles with the plasma membrane in the General protein secretion pathway.
Paul E. O'maille - One of the best experts on this subject based on the ideXlab platform.
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Automating Gene Library synthesis by structure-based combinatorial protein engineering: examples from plant sesquiterpene synthases.
Natural Product Biosynthesis by Microorganisms and Plants Part A, 2012Co-Authors: Melissa Dokarry, Caroline Laurendon, Paul E. O'mailleAbstract:Structure-based combinatorial protein engineering (SCOPE) is a homology-independent recombination method to create multiple crossover Gene libraries by assembling defined combinations of structural elements ranging from single mutations to domains of protein structure. SCOPE was originally inspired by DNA shuffling, which mimics recombination during meiosis, where mutations from parental Genes are "shuffled" to create novel combinations in the resulting progeny. DNA shuffling utilizes sequence identity between parental Genes to mediate template-switching events (the annealing and extension of one parental Gene fragment on another) in PCR reassembly reactions to Generate crossovers and hence recombination between parental Genes. In light of the conservation of protein structure and deGeneracy of sequence, SCOPE was developed to enable the "shuffling" of distantly related Genes with no requirement for sequence identity. The central principle involves the use of oligonucleotides to encode for crossover regions to choreograph template-switching events during PCR assembly of Gene fragments to create chimeric Genes. This approach was initially developed to create libraries of hybrid DNA polymerases from distantly related parents, and later developed to create a combinatorial mutant Library of sesquiterpene synthases to explore the catalytic landscapes underlying the functional divergence of related enzymes. This chapter presents a simplified protocol of SCOPE that can be integrated with different mutaGenesis techniques and is suitable for automation by liquid-handling robots. Two examples are presented to illustrate the application of SCOPE to create Gene libraries using plant sesquiterpene synthases as the model system. In the first example, we outline how to create an active-site Library as a series of complex mixtures of diverse mutants. In the second example, we outline how to create a focused Library as an array of individual clones to distil minimal combinations of functionally important mutations. Through these examples, the principles of the technique are illustrated and the suitability of automating various aspects of the procedure for given applications are discussed.
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Gene Library synthesis by structure-based combinatorial protein engineering.
Methods in Enzymology, 2004Co-Authors: Paul E. O'maille, Ming-daw Tsai, Bryan T. Greenhagen, Joseph Chappell, Joseph P. NoelAbstract:Publisher Summary This chapter discusses the Gene Library synthesis by structure-based combinatorial protein engineering (SCOPE). SCOPE is a process for the synthesis of Gene libraries that lay the Genetic foundation for the exploration of the relationship between structure and function in the encoded proteins. The comparative analysis of both structurally and functionally of protein primary, secondary, and tertiary structure Generates numerous hypotheses to probe the relationship between molecular structure and the ensuing functional readout. SCOPE was applied to create a Library representing all possible combinations of nine point mutations in the terpene cyclase, tobacco 5-epi-aristolochene synthase (TEAS). The location of mutations in the amino acid and nucleotide sequences of TEAS are presented. Over 600 colonies from discrete mixtures, representing about half of the complexity of the TEAS Library, were picked and their sequences determined. It is found that aside from the low-level appearance of wild-type sequence and random mutations likely arising from PCR errors, the actual distribution of mutations in a given mixture was as designed experimentally.